Rotaviruses are a major cause of acute dehydrating diarrhea in infants and young children. Rotavirus disease accounts for 25% to 30% of gastroenteritis deaths in infants and young children in developing countries and approximately 50,000-100,000 hospitalizations of children younger than five years of age in the United States. For this reason, a safe effective vaccine is needed to prevent severe rotavirus disease in infants and young children.
A primary strategy for rotavirus vaccine development has been based on a “Jennerian” approach, which takes advantage of the antigenic relatedness of human and animal rotaviruses and the diminished virulence of animal rotavirus strains in humans. Kapikian et al., in Vaccines 88, Chanock et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 151-159 (1988). Several candidate live oral rotavirus vaccines have been developed using this approach, where an antigenically-related live virus derived from a nonhuman host is used as a vaccine for immunization against its human virus counterpart. Examples of animal rotaviruses that have been used to vaccinate humans include bovine rotavirus strain NCDV (RIT4237, Vesikari et al, Lancet, 2:807-811 (1983)), bovine rotavirus strain WC3 (Clark et al., Am. J. Dis. Child., 140:350-356 (1986)) and rhesus monkey rotavirus (RRV) strain MMU 18006 (U.S. Pat. No. 4,571,385, Kapikian et al., Vaccines 85, eds., Lerner et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 357-367 (1985)).
The protective efficacy among different monovalent bovine and monovalent simian rotavirus vaccines has proved to be variable (Vesikari in Viral Infections of the Gastrointestinal Tract (Kapikian, Ed., Marel Dekker, Inc. pp. 419-442 (1994): Kapikian ibid. pp. 443-470 (1994). Also, high concentrations of bovine rotavirus have been required to produce a satisfactory immune response in humans (107-108 plaque forming units (pfu)) (Vesikari et al., Ped. Inf. Dis. 4:622-625 (1985), Bernstein et al. J. Infect. Dis. 162:1055-1062 (1990)). The variable efficacy of these compositions can in part be attributed to the fact that the target population of two- to five-month old infants characteristically developed a homotypic immune response following vaccination (Kapikian et al., Adv. Exp. Med. Biol., 327:59-69 (1992); Bernstein et al., J. Infect. Dis. 162:1055-1062 (1990); Green et al. J. Infect. Dis. 161:667-679 (1990); and Vesikari, Vaccine. 11:255-261 (1993)).
Clinically relevant human rotaviruses are members of the Group A rotaviruses. These viruses share a common group antigen mediated by VP6, a protein located on the virus intermediate shell. Also, serotype specificity depends on the presence of the VP4 (protease sensitive or P type) and VP7 (glycoprotein or G type) proteins located on the virus outer shell (also often referred to as the virus capsid), both of which independently induce neutralizing antibodies. Kapikian et al. In Virology, Fields, ed. pps. 1353-1404 (1995).
Group A rotaviruses that infect humans have been classified into ten distinct VP7 serotypes by neutralization assays. Amino acid sequence analysis has indicated that within each serotype amino acid identity within two major variable regions was high (85-100%); however, amino acid identity between strains of different serotypes was significantly less (Green et al., Virol. 168:429-433 (1989); Green et al., Virol. 161:153-159 (1987); and Green et al., J. Virol. 62:1819-1823 (1988)). Concordance between relationships among rotaviruses as determined by virus neutralization assay or sequence analysis of VP7 has been demonstrated. Therefore, a reference strain can be routinely used in clinical studies as a representative of rotavirus strains within its serotype.
To achieve protection against each of the four epidemiologically and clinically important G serotypes (VP7) (numbered 1, 2, 3, and 4), the Jennerian approach has been modified by the production of reassortant rotaviruses. Reassortant rotavirus strains were constructed by coinfecting tissue culture cells with a rotavirus of animal origin (i.e., rhesus or bovine rotavirus) and a human rotavirus strain. Reassortant viruses produced during coinfection that contained a single human rotavirus gene encoding VP7 from the human strain and the 10 remaining rotavirus genes from the animal strain were selected by exposing the progeny of the coinfection to a set of monoclonal antibodies directed to the VP7 of the animal strain. (See, for example. U.S. Pat. No. 4,571,385; Midthun et al. J. Clin. Microbiol. 24:822-826 (1986); and Midthun et al. J. Virol. 53:949-954 (1985)).
Studies of human×rhesus rotavirus reassortants and human×bovine reassortants containing the VP7 gene from a human strain have demonstrated that the VP4 neutralization protein of the animal rotavirus parent dominates the immune response in infants vaccinated with these human×animal rotavirus reassortants. This probably reflects the absence of animal rotavirus VP4 antibodies among the antibodies transferred from the mother to the infant in utero. Nevertheless, the immune response to human rotavirus VP7 that is partially blunted by maternally derived VP7 antibodies is sufficient to provide protection and thus VP7 antibodies form the basis of the modified Jennerian approach (Flores et al., J. Clin. Microbiol. 27:512-518 (1989); Perez-Schael et al., J. Clin. Microbiol. 28:553-558 (1990); Flores et al., J. Clin. Microbiol. 31:2439-2445 (1993); Christy et al., J. Infect. Dis. 168:1598-1599 (1993); Clark et al., Vaccine 8:327-332 (1990); Treanor et al., Pediatr. Infect. Dis. J. 14:301-307 (1995); Madore et al. J. Infect. Dis. 166:235-243 (1992); and Clark et al., J. Infect. Dis. 161:1099-1104 (1990).
In studies using a single rhesus rotavirus reassortant bearing a single human rotavirus gene, namely the gene that encodes VP7, it was observed that the protective immunological response of such a reassortant was characteristically homotypic in infants less than six months of age (Green et al., J. Inf. Dis. 161:667-679 (1990)). This observation provided further evidence for the importance of VP7-associated immunity in immunization against rotavirus disease.
The general experience with monovalent and quadrivalent human×rhesus rotavirus reassortant vaccines has been that a transient low-level febrile episode occurs in about one-third of young infants 3 to 4 days after vaccination. Bernstein et al., JAMA 273:1191-1196 (1995); Flores et al., Lancet 336:330-334 (1995); Perez-Schael et al., J. Clin. Microbiol. 28:553-558 (1990); Flores et al., J. Clin. Microbiol. 31:2439-2445 (1990); Halsey et al., J. Infect Dis. 158:1261-1267 (1988); Taniguichi et al., J. Clin. Microbiol. 29:483-487 (1991); Simasathien et al., Pediatr. Infect. Dis. J. 13:590-596 (1994); Madore et al., J. Infect. Dis. 166:235-243 (1992); and Joensuu et al., Lancet 350:1205-1209 (1997).
Results of studies in humans with bovine rotavirus strains NCDV and WC3 (VP7 serotype 6) indicate that these particular bovine rotavirus strains do not appear to cause fever or other reactions. It should be noted that serotype 6 VP7 is not known to be present on human rotaviruses that are important in human rotavirus disease. Also, a bovine rotavirus was not found to be as immunogenic as the rhesus rotavirus when administered to humans. The bovine rotavirus strain NCDV (RIT4237 vaccine) has been evaluated in more than five efficacy trials in infants and young children. In these trials, the bovine RIT4237 vaccine was administered at a dose range of 107.8 to 108.3 tissue culture infectious doses 50 (TCID50), with the usual dosage exceeding 108.0 TCID50. Also, in a dose-response study, Vesikari et al., Ped. Infect. Dis., 4:622-625 (1985)) observed that 15% (2/13) of four- to six-month old infants developed a homotypic antibody response when the vaccine was administered at a dose of 106.3 TCID50; 71% (10/14) when administered at a dose of 107.2 TCID50, and 100% when administered at a dose of 108.3 TCID50. Thus, the dose for this bovine rotavirus strain that produced an optimal immunogenicity was determined to be in the range of 108.0 TCID50.
In a direct comparison of the infectivity and immunogenicity of rhesus rotavirus and bovine rotavirus in humans, 105 plaque forming units (pfu) of rhesus rotavirus (RRV vaccine) or 108.3 pfu of RIT4237 was administered to children six to eight months of age. (Vesikari et al., J. Infect. Dis. 153:832-839 (1986)). The RRV vaccine induced a homotypic neutralizing antibody response in 81% of vaccinees, whereas the two thousand fold greater dose of the bovine RIT4237 vaccine induced homotypic neutralizing antibodies in only 45% of vaccinees, which was a statistically significant difference.
Efficacy trials were also conducted with the WC3 bovine rotavirus strain. In these trials, the WC3 strain was administered to infants and young children at a dose range of 107.0 to 107.3 pfu. (Clark et al., Am. J. Dis. Child. 140:350-356 (1986)). Although data regarding the dose required for significant immunogenicity was not provided, Clark et al. noted that the WC3 strain appears to possess safety characteristics similar to those of RIT4237, yet was immunogenic at a dose at least five fold less than that used with bovine RIT4237, though this immunogenicity still required a dose that was considerably greater than that of rhesus rotavirus vaccine.
The WC3 rotavirus strain has been used as one of the parent strains for generating reassortants with various human rotavirus strains. (Clark et al., J. Infect. Dis. (suppl.) 174:73-80 (1996)). In one efficacy trial, 1073 pfu of a monovalent reassortant of WC3 and a human rotavirus VP7 serotype 1 was administered on a three dose schedule to infants and young children. (Treanor et al., Ped. Inf. Dis. J. 14:301-307 (1995)). Immunogenicity data was not reported for this trial. In another efficacy study, a quadrivalent formulation was used which contained three human VP7 reassortants of bovine rotavirus WC3 with a human rotavirus VP7 serotype of 1, 2, or 3 and as a fourth component, a human x bovine reassortant bearing a human rotavirus VP4 protein with the remaining genes derived from the bovine rotavirus WC3. Each of the three VP7 reassortants was used at a dose of 107.0 pfu, while the VP4 reassortant was administered at a dosage of 5×106.0 pfu. (Clark et al., Arch. Virol. (suppl.) 12:187-198 (1996); Clark et al. J. Infect. Dis. (suppl.) 174:73-80 (1996); Vesikari et al. Arch. Virol. (suppl.) 12:177-186 (1996)). Immunogenicity data for this trial also was not reported, but these studies indicate that to characteristically produce a protective response similar to that obtained with the rhesus rotavirus or human×rhesus reassortant vaccines a dosage of 107 to 108.3 pfu was required. (Clark et al. Arch. Virol. (suppl.) 12:187-198 (1996); Vesikari et al. Arch. Virol. (suppl.) 12:177-186 (1996)). This dosage is 10 to 100 times higher than that for the rhesus rotavirus and human×rhesus rotavirus reassortant vaccine compositions.
Multivalent rotavirus vaccine compositions have been developed. In particular, three human×rhesus rotavirus reassortants representing human serotypes 1, 2 and 4 have been combined with a rhesus rotavirus strain (RRV) (the latter sharing neutralization specificity with human serotype 3) to form a quadrivalent vaccine composition (Perez-Schael et al., J. Clin. Microbiol. 28:553-558 (1990), Flores et al., J. Clin. Microbiol. 31:2439-2445 (1993)). As with the monovalent rhesus rotavirus, the human×rhesus reassortant rotavirus vaccine compositions were found to produce a transient low level febrile condition in approximately 15% to 33% of the infants vaccinated (Perez-Schael et al. supra). This transient febrile episode or condition, although generally considered acceptable by the parents and health care providers of the clinical trial, could possibly be a deterrent in certain situations, such as, in premature infants who may have low levels of passively acquired maternal antibodies to rotavirus and the like.
Although the animal rotavirus-based rotavirus vaccine composition presently licensed by the United States Food and Drug Administration provides an important level of protection in humans against rotavirus infection, a multivalent vaccine composition with both high infectivity and which produce little or no febrile response is desirable, especially for certain clinical situations. Surprisingly, the present invention fulfills these and other related needs.